Model Systems and Treatment Regimes for Treatment of Neurological Disease

ABSTRACT

The invention provides animal models and clinical trials for assessing agents for potential use in treating and effecting prophylaxis stroke and other neurological diseases, particularly those mediated at least in part by excitoxitity. The invention also provides preferred dosage and infusion regimes and pharmaceutical compositions for clinical application of such agents.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a nonprovisional and claims the benefit ofU.S. Provisional App. No. 61/185,989 filed Jun. 10, 2009.

BACKGROUND OF THE INVENTION

Early work on excitotoxicity suggested that stroke and neurotrauma mightbe treated using drugs that block glutamate receptors (Albers, Archivesin Neurology 49:418-420 (1992); Albers Ann Neurol 25:398-403 (1989)).Although early tests in vitro and in animal models were promising,unfortunately, all clinical stroke trials of glutamate antagonists todate have shown no benefit, and even toxic side effects (Davis et al.,Lancet 349:32 (1997); Morris et al., J Neurosurg 91:737-743 (1999);Davis et al., Stroke 31:347-354 (2000); Ikonomidou et al., Proc NatlAcad Sci USA 97:12885-12890 (2000); Lees et al., Lancet 355:1949-1954(2000)). A likely explanation is that the negative consequences ofadministering agents that inhibit excitatory neurotransmission in theCNS had outweighed their utility as neuroprotectants (Ikonomidou andTurski, Lancet Neurology 383-386 (2002). Glutamatergic signalling isrequired for CNS function and therefore, blocking essential excitatoryneurotransmission has negative consequences (Ikonomidou, Biochem.Pharmacol. 62:401-405 (2001)). Thus, a more sophisticated approach totreating neuronal death is required to bypass the negative consequencesof blocking glutamate receptors.

One of the present inventors has reported that postsynaptic density-95protein (PSD-95) couples NMDARs to pathways mediating excitotoxicity andischemic brain damage (Aarts et al., Science 298, 846-850 (2002)). Thiscoupling was disrupted by transducing neurons with peptides that bind tomodular domains on either side of the PSD-95/NMDAR interaction complex.This treatment attenuated downstream NMDAR signaling without blockingNMDAR activity, protected cultured cortical neurons from excitotoxicinsults and reduced cerebral infarction volume in rats subjected totransient focal cerebral ischemia.

SUMMARY OF THE INVENTION

The invention provides methods of treating or effecting prophylaxis ofdisease mediated by excitotoxicity, comprising administering to asubject having or at risk of the disease a pharmacological agent thatinhibits binding of PSD-95 to NMDAR 2B and/or of PSD-95 to nNOS, whereinwhen the agent is a peptide having the amino acid sequenceYGRKKRRQRRRKLSSIESDV (SEQ ID NO:6), the dose is 2-3 mg/kg, and if theagent is other than the peptide having the amino acid sequenceYGRKKRRQRRRKLSSIESDV, the dose delivers the equivalent effectiveconcentration of the agent to 2-3 mg/kg of the peptide having the aminoacid sequence YGRKKRRQRRRKLSSIESDV. In some methods, the agent is thepeptide having the amino acid sequence YGRKKRRQRRRKLSSIESDV and the doseis 2.6 mg/kg. In some methods, the dose is administered once per episodeof the disease. In some methods, the dose is administeredwithout-co-administration of an anti-inflammatory agent.

The invention also provides methods of treating or effecting prophylaxisof a disease mediated by excitotoxicity, comprising administering to asubject having or at risk of the disease a pharmacological agent thatinhibits binding of PSD-95 to NMDAR 2B and/or PSD-95 to nNOS, whereinthe agent is linked to an internalization peptide, and the agent isadministered by intravenous infusion over a period of 5-15 minutes. Insome methods, the agent linked to the internationalization peptide isthe peptide having the amino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ IDNO:6) and the period is 5 minutes. In some methods, the dose of thepharmacological agent is greater than 1 mg/kg. In some methods, the doseof the pharmacological agent is 2-3 mg/kg. In some methods, the dose ofthe pharmacological agent is 2.6 mg/kg. In some methods, the dose is upto 50 mg/kg, provided that if the dose is greater than 3 mg/kg the doseis co-administered with an anti-inflammatory. In some methods, isadministered without co-administration of an anti-inflammatory agent. Insome methods, the subject has a stroke. In some methods, the subject isundergoing surgery, optionally, endovascular surgery to treat ananeurysm.

The invention further provides methods of inhibiting ischemic damagefrom neurosurgery or endovascular surgery (whether or not in the CNS),comprising administering an effective regime of an agent that inhibitsbinding of PSD-95 to NMDAR 2B to a patient undergoing neurosurgery. Insome methods, the neurosurgery is diagnostic angiography of the brain.In some methods, the neurosurgery is endovascular surgery to treat ananeurysm. In some methods, the agent is administered before theendovascular surgery. In some methods, the agent is administered within1 hour of completing endovascular surgery. In some methods, theendovascular surgery comprising inserting a coil into the aneurysm. Insome methods, the endovascular surgery comprises inserting a stent intothe vessel subject to the aneurysm. In some methods, the endovascularsurgery comprises inserting a microcatheter.

The invention provides methods of performing a clinical trial on apharmacological agent comprising administering the pharmacological agentto a population of patients undergoing endovascular surgery for a brainaneurysm, comparing the frequency of a damaging effect of the surgery inthe patients compared with control patients undergoing the endovascularsurgery without the pharmacological agent, to determine whether thepharmacological agent reduces the damaging effect. In some methods, thedamaging effect is assessed by the number and/or size of cerebralinfarctions. Some methods further comprise testing the pharmacologicalagent in an animal model of stroke. Some methods further comprisetesting the pharmacological agent in a human patient having a stroke.Some methods further comprise labeling the pharmacological agent fortreatment of stroke. In some methods, the pharmacological agent inhibitsbinding of PSD95 to NMDAR 2B and/or PSD-95 to nNOS.

The invention further provides methods of testing a pharmacologicalagent. Such methods involve: (a) inserting particles into a cerebralblood vessel of a primate; (b) administering the pharmacological agentto the primate; and (c) comparing a damaging effect in the primatecompared to a control primate not treated with the compound. In somemethods, the damaging effect is assessed by number and/or size ofinfarctions. In some methods, the infarctions are determined by MRI orCAT scanning. In some methods, the damaging effect is assessed by abehavioral symptom of the primate compared with the control primate.Some methods further comprise repeating steps (a)-(c) on the sameprimate after a recovery period provided the agent tested on repeatingsteps (a)-(c) may or may not be the same as when steps (a)-(c) werepreviously preformed. Some methods further comprise repeating steps(a)-(c) except that the control primate receives the agent and theanimal previously receiving the agent is the control animal. In somemethods, the agent inhibits binding of PSD-95 to NMDAR 2B and/or PSD-95to nNOS. Some methods further comprise administering the agent to ahuman stroke subject. Some methods further comprise administering theagent to a human subject undergoing endovascular surgery for ananeurysm. In some methods, the agent is administered after insertingparticles into the cerebral blood vessel. In some methods, the agent isadministered before inserting particles into the cerebral blood vessel.In some methods, the particles are 100 micron polystyrene spheres. Insome methods, 20 spheres are administered to the primate. In somemethods, the primate is a macaque.

The invention further provides a lyophilized composition comprising thepeptide having the amino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ IDNO:6) lyophilized from a solution of the peptide in normal salineprepared under GMP conditions.

The invention further provides a method of storing a pharmaceuticalcomposition comprising storing a pharmaceutical composition comprising apeptide having the amino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ IDNO:6) at concentration of 10-30 mg/ml in normal saline under GMPconditions at below 0° C. for a period of at least two years. Optionallythe concentration is 18-20 mg/ml. Some methods further compriseadministering the pharmaceutical composition to a patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diffusion-weighted MRI scan of infarctions 24 hr postinjection of spheres in a control animal compared with a human havingreceived endovascular surgery to insert a coil to repair an aneurysm.The upper portion shows diffusion defects 48 hours post aneurysm coilingin a 44 year old female human. The lower portion shows diffusion defects24 hours post injection of 20×100 um poly-styrene microspheres in a malecynomolgus macaque.

FIG. 2 compares the number and volume of MRI-visible infarctions intreated and control animals.

FIG. 3 shows similar data to FIG. 2 for infarctions in the cortex.

FIG. 4 shows changes in blood pressure following administration ofTat-NR2B9c at 50 mg/kg and an infusion time of 3 min or 60 min.

FIG. 5 shows infarction size measured 24 hr post treatment wassignificantly reduced relative to saline placebo with the 5 min infusionbut not with the one hour infusion of Tat-NR2B9c at 7.6 mg/kg (rightmostcolumn).

DEFINITIONS

A “chimeric peptide” means a peptide having two component peptides notnaturally associated with one another joined to one another as a fusionprotein or by chemical linkage.

A “fusion” protein or polypeptide refers to a composite polypeptide,i.e., a single contiguous amino acid sequence, made up of sequences fromtwo (or more) distinct, heterologous polypeptides which are not normallyfused together in a single polypeptide sequence.

The term “PDZ domain” refers to a modular protein domain of about 90amino acids, characterized by significant sequence identity (e.g., atleast 60%) to the brain synaptic protein PSD-95, the Drosophila septatejunction protein Discs-Large (DLG), and the epithelial tight junctionprotein ZO1 (Z01). PDZ domains are also known as Discs-Large homologyrepeats (“DHRs”) and GLGF repeats. PDZ domains generally appear tomaintain a core consensus sequence (Doyle, D. A., 1996, Cell 85:1067-76). Exemplary PDZ domain-containing proteins and PDZ domainsequences disclosed in U.S. application Ser. No. 10/714,537, which isherein incorporated by reference in its entirety.

The term “PL protein” or “PDZ Ligand protein” refers to a naturallyoccurring protein that forms a molecular complex with a PDZ-domain, orto a protein whose carboxy-terminus, when expressed separately from thefull length protein (e.g., as a peptide fragment of 3-25 residues, e.g.3, 4, 5, 8, 9, 10, 12, 14 or 16 residues), forms such a molecularcomplex. The molecular complex can be observed in vitro using the “Aassay” or “G assay” described, e.g., in U.S. application Ser. No.10/714,537, or in vivo.

The term “NMDA receptor,” or “NMDAR,” refers to a membrane associatedprotein that is known to interact with NMDA including the varioussubunit forms described below. Such receptors can be human or non-human(e.g., mouse, rat, rabbit, monkey).

A “PL motif” refers to the amino acid sequence of the C-terminus of a PLprotein (e.g., the C-terminal 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or25 contiguous residues) (“C-terminal PL sequence”) or to an internalsequence known to bind a PDZ domain (“internal PL sequence”).

A “PL peptide” is a peptide of comprising or consisting of, or otherwisebased on, a PL motif that specifically binds to a PDZ domain.

The terms “isolated” or “purified” means that the object species (e.g.,a peptide) has been purified from contaminants that are present in asample, such as a sample obtained from natural sources that contain theobject species. If an object species is isolated or purified it is thepredominant macromolecular (e.g., polypeptide) species present in asample (i.e., on a molar basis it is more abundant than any otherindividual species in the composition), and preferably the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, an isolated, purified orsubstantially pure composition comprises more than 80 to 90 percent ofall macromolecular species present in a composition. Most preferably,the object species is purified to essential homogeneity (i.e.,contaminant species cannot be detected in the composition byconventional detection methods), wherein the composition consistsessentially of a single macromolecular species. The term isolated orpurified does not necessarily exclude the presence of other componentsintended to act in combination with an isolated species. For example, aninternalization peptide can be described as isolated notwithstandingthat it is linked to an active peptide.

A “peptidomimetic” refers to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics of apeptide consisting of natural amino acids. The peptidomimetic cancontain entirely synthetic, non-natural analogues of amino acids, or canbe a chimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The peptidomimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or inhibitory or binding activity. Polypeptidemimetic compositions can contain any combination of nonnaturalstructural components, which are typically from three structural groups:a) residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., to induce or stabilize a secondary structure, e.g., abeta turn, gamma turn, beta sheet, alpha helix conformation, and thelike. In a peptidomimetic of a chimeric peptide comprising an activepeptide and an internalization peptide, either the active moiety or theinternalization moiety or both can be a peptidomimetic.

The term “specific binding” refers to binding between two molecules, forexample, a ligand and a receptor, characterized by the ability of amolecule (ligand) to associate with another specific molecule (receptor)even in the presence of many other diverse molecules, i.e., to showpreferential binding of one molecule for another in a heterogeneousmixture of molecules. Specific binding of a ligand to a receptor is alsoevidenced by reduced binding of a detectably labeled ligand to thereceptor in the presence of excess unlabeled ligand (i.e., a bindingcompetition assay).

Excitotoxicity is the pathological process by which neurons are damagedand killed by the overactivation of receptors for the excitatoryneurotransmitter glutamate, such as the NMDA receptors, e.g., NMDAR 2B.

The term “subject” or “patient” includes humans and veterinary animals,such as mammals.

The term “agent” includes any compound including compounds with orwithout pharmaceutical activity, natural compounds, synthetic compounds,small molecules, peptides and peptidomimetics.

The term “pharmacologic agent” means an agent having a pharmacologicalactivity. Pharmacological agents include compounds that are known drugs,compounds for which pharmacological activity has been identified butwhich are undergoing further therapeutic evaluation in animal models orclinical trials. A chimeric agent comprises a pharmacologic agent linkedto an internalization peptide. An agent can be described as havingpharmacological activity if it exhibits an activity in a screeningsystem that indicates that the active agent is or may be useful in theprophylaxis or treatment of a disease. The screening system can be invitro, cellular, animal or human. Agents can be described as havingpharmacological activity notwithstanding that further testing may berequired to establish actual prophylactic or therapeutic utility intreatment of a disease.

A tat peptide means a peptide comprising or consisting of GRKKRRQRRR(SEQ ID NO:1), in which no more than 5 residues are deleted, substitutedor inserted within the sequence, which retains the capacity tofacilitate uptake of a linked peptide or other agent into cells.Preferably any amino acid changes are conservative substitutions.Preferably, any substitutions, deletions or internal insertions in theaggregate leave the peptide with a net cationic charge, preferablysimilar to that of the above sequence. The amino acids of a tat peptidecan be derivatized with biotin or similar molecule to reduce aninflammatory response.

Co-administration of a pharmacological agents linked to aninternalization peptide and an anti-inflammatory agent means that thetwo agents are administered sufficiently proximately in time that theanti-inflammatory agent can inhibit an inflammatory response inducibleby the internationalization peptide.

Statistically significant refers to a p-value that is <0.05, preferably<0.01 and most preferably <0.001.

An episode of a disease means a period when signs and/or symptoms of thedisease are present interspersed by flanked by longer periods in whichthe signs and/or symptoms or absent or present to a lesser extent.

DETAILED DESCRIPTION OF THE INVENTION I. General

The invention provides animal models and clinical trials for assessingagents for potential use in treating or effecting prophylaxis for strokeand other neurological diseases, particularly those mediated at least inpart by excitotoxicity. The invention also provides preferred dosage andinfusion regimes and pharmaceutical compositions for clinicalapplication of such agents.

II. Agents for Treating Disease

Although any agents can be screened for efficacy in the animal models orclinical trials described below, including agents that have failedprevious clinical trials for stroke, a preferred class of agentsinhibits interactions between PSD-95 and one or more NMDARs. Such agentsare useful for reducing damaging effects of stroke and otherneurological conditions mediated at least in part by NMDARexcitotoxicity. Such agents include peptides having an amino acidsequence including or based on the PL motif of a NMDA Receptor or PDZdomain of PSD95. Such peptides can also inhibit interactions betweenPSD-95 and nNOS and other glutamate receptors (e.g., kainite receptorsor AMPA receptors), such as KV1-4 and GluR6. Preferred peptides inhibitinteraction between PDZ domains 1 and 2 of postsynaptic density-95protein (PSD-95) (human amino acid sequence provided by Stathakism,Genomics 44(1):71-82 (1997)) and the C-terminal PL sequence of one ormore NMDA Receptor 2 subunits including the NR2B subunit of the neuronalN-methyl-D-aspartate receptor (Mandich et al., Genomics 22, 216-8(1994)). NMDAR2B has GenBank ID 4099612, a C-terminal 20 amino acidsFNGSSNGHVYEKLSSIESDV (SEQ ID NO:11) and a PL motif ESDV (SEQ ID NO:12).Preferred peptides inhibit the human forms of PSD-95 and human NMDARreceptors. However, inhibition can also be shown from species variantsof the proteins. A list of NMDA and glutamate receptors that can be usedappears below:

TABLE 1 NMDA Receptors With PL Sequences C-terminal internal NameGI or Acc # C-terminal 20 mer sequence 4 mer sequence PL? PL ID NMDAR1  307302 HPTDITGPLNLSDPSVSTVV STVV X AA216 (SEQ ID NO: 13)(SEQ ID NO: 27) NMDAR1-1   292282 HPTDITGPLNLSDPSVSTVV STVV X AA216(SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1-4   472845 HPTDITGPLNLSDPSVSTVVSTVV X AA216 (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1-3b  2343286HPTDITGPLNLSDPSVSTVV STVV X AA216 (SEQ ID NO: 13) (SEQ ID NO: 27)NMDAR1-4b  2343288 HPTDITGPLNLSDPSVSTVV STVV X AA216 (SEQ ID NO: 13)(SEQ ID NO: 27) NMDAR1-2 11038634 RRAIEREEGQLQLCSRHRES HRES(SEQ ID NO: 14) (SEQ ID NO: 28) NMDAR1-3 11038636 RRAIEREEGQGQLCSRHRESHRES (SEQ ID NO: 14) (SEQ ID NO: 28) NMDAR2C  6006004TQGFPGPCTWRRISSLESEV ESEV X AA180 (SEQ ID NO: 15) (SEQ ID NO: 29) NMDAR3  560546 FNGSSNGVHYEKLSSIESDV ESDV X AA34.1 (SEQ ID NO: 11)(SEQ ID NO: 12) NMDAR3A 17530176 AVSRKTELEEYQRTSRTCES TCES(SEQ ID NO: 16) (SEQ ID NO: 30) NMDAR2B  4099612 FNGSSNGHVYEKLSSIESDVESDV X (SEQ ID NO: 11) (SEQ ID NO: 12) NMDAR2A   558748LNSCSNRRVYKKMPSIESDV ESDV X AA34.2 (SEQ ID NO: 17) (SEQ ID NO: 12)NMDAR2D  4504130 GGDLGTRRGSAHFSSLESEV ESEV X (SEQ ID NO: 18)(SEQ ID NO: 29) Glutamate AF009014 QPTPTLGLNLGNDPDRGTSI GTSI Xreceptor delta 2 (SEQ ID NO: 19) (SEQ ID NO: 31) Glutamate   I28953MQSIPCMSHSSGMPLGATGL ATGL X receptor 1 (SEQ ID NO: 20) (SEQ ID NO: 32)Glutamate   L20814 QNFATYKEGYNVYGIESVKI SVKI X receptor 2(SEQ ID NO: 21) (SEQ ID NO: 33) Glutamate AF167332 QNYATYREGYNVYGTESVKISVKI X receptor 3 (SEQ ID NO: 22) (SEQ ID NO: 33) Glutamate   U16129HTGTAIRQSSGLAVIASDLP SDLP receptor 4 (SEQ ID NO: 23) (SEQ ID NO: 34)Glutamate   U16125 SFTSILTCHQRRTQRKETVA ETVA X receptor 5(SEQ ID NO: 24) (SEQ ID NO: 35) Glutamate   U16126 EVINMHTFNDRRLPGKETMAETMA X receptor 6 (SEQ ID NO: 25) (SEQ ID NO: 36)

Some peptides inhibit interactions between PSD-95 and multiple NMDARsubunits. In such instances, use of the peptide does not necessarilyrequire an understanding of the respective contributions of thedifferent NMDARs to excitatory neurotransmission. Other peptides arespecific for a single NMDAR.

Peptides can include or be based on a PL motif from the C-terminus ofany of the above subunits and have an amino acid sequence comprising[S/T]-X-[V/L]. This sequence preferably occurs at the C-terminus of thepeptides of the invention. Preferred peptides have an amino acidsequence comprising [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:38) attheir C-terminus. Exemplary peptides comprise: ESDV (SEQ ID NO:12), ESEV(SEQ ID NO:29), ETDV (SEQ ID NO:39), ETEV (SEQ ID NO:40), DTDV (SEQ IDNO:41), and DTEV (SEQ ID NO:42) as the C-terminal amino acids. Twoparticularly preferred peptides are KLSSIESDV (SEQ ID NO:5), andKLSSIETDV (SEQ ID NO:43). Such peptides usually have 3-25 amino acids(without an internalization peptide), peptide lengths of 5-10 aminoacids, and particularly 9 amino acids (also without an internalizationpeptide) are preferred. In some such peptides, all amino acids are fromthe C-terminus of an NMDA receptor (not including amino acids from aninternalization peptide).

Other peptides that inhibit interactions between PDS95 and NDMARsinclude peptides from PDZ domain 1 and/or 2 of PSD-95 or a subfragmentof any of these that inhibits interactions between PSD-95 and an NMDAreceptor, such as NMDA 2B. Such active peptides comprise at least 50,60, 70, 80 or 90 amino acids from PDZ domain 1 and/or PDZ domain 2 ofPSD-95, which occur within approximately amino acids 65-248 of PSD-95provided by Stathakism, Genomics 44(1):71-82 (1997) (human sequence) orNP_(—)031890.1, GI:6681195 (mouse sequence) or corresponding regions ofother species variants.

Peptides and peptidomimetics of the invention can contain modified aminoacid residues for example, residues that are N-alkylated. N-terminalalkyl modifications can include e.g., N-Methyl, N-Ethyl, N-Propyl,N-Butyl, N-Cyclohexylmethyl, N-Cyclyhexylethyl, N-Benzyl, N-Phenylethyl,N-phenylpropyl, N-(3,4-Dichlorophenyl)propyl,N-(3,4-Difluorophenyl)propyl, and N-(Naphthalene-2-yl)ethyl).

Bach, J. Med. Chem. 51, 6450-6459 (2008) and WO 2010/004003 hasdescribed a series of analogs of NMDAR 2B 9c. PDZ-binding activity isexhibited by peptides having only three C-terminal amino acids (SDV).Bach also reports analogs having an amino acid sequence comprising orconsisting of YtSXV (SEQ ID NO:68), wherein t and S are alternativeamino acids, Y is selected from among E, Q, and A, or an analoguethereof, X is selected from among A, Q, D, N,N-Me-A, N-Me-Q, N-Me-D, andN-Me-N or an analogue thereof. Optionally the peptide is N-alkylated inposition P3 position (third amino acid from C-terminus, i.e. positionoccupied by tS). The peptide can be N-alkylated with a cyclohexane oraromatic substituent, and further comprises a spacer group between thesubstituent and the terminal amino group of the peptide or peptideanalogue, wherein the spacer is an alkyl group, preferably selected fromamong methylene, ethylene, propylene and butylene. The aromaticsubstituent can be a naphthalen-2-yl moiety or an aromatic ringsubstituted with one or two halogen and/or alkyl group.

Other modifications can also be incorporated without adversely affectingthe activity and these include substitution of one or more of the aminoacids in the natural L-isomeric form with amino acids in the D-isomericform. Thus, any amino acid naturally occurring in the L-configuration(which can also be referred to as the R or S, depending upon thestructure of the chemical entity) can be replaced with the amino acid ofthe same chemical structural type or a peptidomimetic, but of theopposite chirality, generally referred to as the D-amino acid, but whichcan additionally be referred to as the R- or S-form. Thus, apeptidomimetic may include 1, 2, 3, 4, 5, at least 50%, or all D-aminoacid resides. A peptidomimetic containing some or all D residues issometimes referred to an “inverso” peptide.

Peptidomimetics also include retro peptides. A retro peptide has areverse amino acid sequence. Peptidomimetics also include retro inversopeptides in which the order of amino acids is reversed from so theoriginally C-terminal amino acid appears at the N-terminus and D-aminoacids are used in place of L-amino acids. WO 2008/014917 describes aretro-inverso analog of Tat-NR2B9c having the amino acid sequencevdseisslk-rrrqrrkkrgyin (SEQ ID NO:69) (lower case letters indicating Damino acids), and reports it to be effective inhibiting cerebralischemia. Another effect peptide described herein is Rv-Tat-NR2B9c(RRRQRRKKRGYKLSSIESDV SEQ ID NO:70).

A linker, e.g., a polyethylene glycol linker, can be used to dimerizethe active moiety of the peptide or the peptidomimetic to enhance itsaffinity and selectivity towards proteins containing tandem PDZ domains.See e.g., Bach et al., (2009) Angew. Chem. Int. Ed. 48:9685-9689 and WO2010/004003. A PL motif-containing peptide is preferably dimerized viajoining the N-termini of two such molecules, leaving the C-termini free.Bach further reports that a pentamer peptide IESDV (SEQ ID NO:71) fromthe C-terminus of NMDAR 2B was effective in inhibiting binding of NMDAR2B to PSD95. Optionally, about 2-10 copies of a PEG can be joined intandem as a linker.

Appropriate pharmacological activity of peptides, peptidomimetics orother agent can be confirmed if desired, using previously described ratmodels of stroke before testing in the primate and clinical trialsdescribed in the present application. Peptides or peptidomimetics canalso be screened for capacity to inhibit interactions between PSD-95 andNMDAR 2B using assays described in e.g., US 20050059597, which isincorporated by reference. Useful peptides typically have 1050 values ofless than 50 μM, 25 μM, 10 μM, 0.1 μM or 0.01 μM in such an assay.Preferred peptides typically have an 1050 value of between 0.001-1 μM,and more preferably 0.05-0.5 or 0.05 to 0.1 μM. When a peptide or otheragent is characterized as inhibiting binding of one interaction, e.g.,PSD-95 interaction to NMDAR2B, such description does not exclude thatthe peptide or agent also inhibits another interaction, for example,inhibition of PSD-95 binding to nNOS.

Peptides such as those just described can optionally be derivatized(e.g., acetylated, phosphorylated and/or glycosylated) to improve thebinding affinity of the inhibitor, to improve the ability of theinhibitor to be transported across a cell membrane or to improvestability. As a specific example, for inhibitors in which the thirdresidue from the C-terminus is S or T, this residue can bephosphorylated before use of the peptide.

Pharmacological agents also include small molecules that inhibitinteractions between PSD95 and NMDAR 2B, and/or other interactionsdescribed above. Suitable small-molecule inhibitors are described inWO/2009/006611. An exemplary class of suitable compounds are of theformula:

wherein R¹ is a member selected from the group consisting of cyclohexylsubstituted with 0-4 R⁷, phenyl substituted with 0-4 R⁷,—(CH₂)_(u)—(CHR⁸R⁹), a branched C₁₋₆ alkyl (isopropyl, isobutyl,1-isopropyl-2-methyl-butyl, 1 ethyl-propyl), and—NH—C(O)—(CR¹⁰R¹¹)_(v)H;

each R⁷ is independently a member selected from the group consisting ofC₁₋₆ alkyl, C₁₋₆ alkoxy, —C(O)R¹², OH, COOH, —NO, N-substituted indolineand a cell membrane translocation peptide;

each R⁸ and R⁹ is independently selected from the group consisting of H,OH, cyclohexane, cyclopentane, phenyl, substituted phenyl andcyclopentadiene;

each R¹⁰ and R¹¹ is independently selected from the group consisting ofH, cyclohexane, phenyl and a cell membrane translocation peptide;

R¹² is a member selected from the group consisting of C₁₋₆ alkyl andaryl; and each of u and v are independently from 0 to 20;

wherein one of R², R³, R⁴, R⁵ and R⁶ is —COOH, and wherein the remainderof R², R³, R⁴, R⁵ and R⁶ are each independently selected from the groupconsisting of F, H, OCH₃ and CH₃.

One such compound is 0620-0057, the structure of which is:

A pharmacological agent can be linked to an internalization peptide tofacilitate uptake into cells and/or across the blood brain barrier.Internalization peptides are a well-known class of relatively shortpeptides that allow many cellular or viral proteins to traversemembranes. Internalization peptides, also known as cell membranetransduction peptides or cell penetrating peptides can have e.g., 5-30amino acids. Such peptides typically have a cationic charge from anabove normal representation (relative to proteins in general) ofarginine and/or lysine residues that is believed to facilitate theirpassage across membranes. Some such peptides have at least 5, 6, 7 or 8arginine and/or lysine residues. Examples include the antennapediaprotein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (and variantsthereof), the tat protein of human immunodeficiency virus, the proteinVP22, the product of the UL49 gene of herpes simplex virus type 1,Penetratin, SynB1 and 3, Transportan, Amphipathic, gp41NLS, polyArg, andseveral plant and bacterial protein toxins, such as ricin, abrin,modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat labiletoxins, and Pseudomonas aeruginosa exotoxin A (ETA). Other examples aredescribed in the following references (Temsamani, Drug Discovery Today,9(23):1012-1019, 2004; De Coupade, Biochem J., 390:407-418, 2005; SaalikBioconjugate Chem. 15: 1246-1253, 2004; Zhao, Medicinal Research Reviews24(1):1-12, 2004; Deshayes, Cellular and Molecular Life Sciences62:1839-49, 2005) (all incorporated by reference).

A preferred internalization peptide is tat from the HIV virus. A tatpeptide reported in previous work comprises or consists of the standardamino acid sequence YGRKKRRQRRR (SEQ ID NO:2) found in HIV Tat protein.If additional residues flanking such a tat motif are present (beside thepharmacological agent) the residues can be for example natural aminoacids flanking this segment from a tat protein, spacer or linker aminoacids of a kind typically used to join two peptide domains, e.g., gly(ser)₄ (SEQ ID NO:44), TGEKP (SEQ ID NO:45), GGRRGGGS (SEQ ID NO:46), orLRQRDGERP (SEQ ID NO:47) (see, e.g., Tang et al. (1996), J. Biol. Chem.271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11, 405-410)), orcan be any other amino acids that do not significantly reduce capacityto confer uptake of the variant without the flanking residues.Preferably, the number of flanking amino acids other than an activepeptide does not exceed ten on either side of YGRKKRRQRRR (SEQ ID NO:2).One suitable tat peptide comprising additional amino acid residuesflanking the C-terminus of YGRKKRRQRRR (SEQ ID NO:2) is YGRKKRRQRRRPQ(SEQ ID NO:48). However, preferably, no flanking amino acids arepresent. Other tat peptides that can be used include GRKKRRQRRRPQ (SEQID NO:4) and GRKKRRQRRRP (SEQ ID NO:72).

Variants of the above tat peptide having reduced capacity to bind toN-type calcium channels are described by WO/2008/109010. Such variantscan comprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ IDNO:49), in which X is an amino acid other than Y or nothing (in whichcase G is a free N-terminal residue). A preferred tat peptide has theN-terminal Y residue substituted with F. Thus, a tat peptide comprisingor consisting of FGRKKRRQRRR (SEQ ID NO:3) is preferred. Anotherpreferred variant tat peptide consists of GRKKRRQRRR (SEQ ID NO:1).Another preferred tat peptide comprises or consists of RRRQRRKKRG (aminoacids 1-11 of SEQ ID NO:70). Other tat derived peptides that facilitateuptake of a pharmacological agent without inhibiting N-type calciumchannels include those presented in Table 2 below.

TABLE 2 X-FGRKKRRQRRR (F-Tat) (SEQ ID NO: 3) X-GKKKKKQKKK(SEQ ID NO: 50) X-RKKRRQRRR (SEQ ID NO: 51) X-GAKKRRQRRR (SEQ ID NO: 52)X-AKKRRQRRR (SEQ ID NO: 53) X-GRKARRQRRR (SEQ ID NO: 54) X-RKARRQRRR(SEQ ID NO: 55) X-GRKKARQRRR (SEQ ID NO: 56) X-RKKARQRRR (SEQ ID NO: 57)X-GRKKRRQARR (SEQ ID NO: 58) X-RKKRRQARR (SEQ ID NO: 59) X-GRKKRRQRAR(SEQ ID NO: 60) X-RKKRRQRAR (SEQ ID NO: 61) X-RRPRRPRRPRR(SEQ ID NO: 62) X-RRARRARRARR (SEQ ID NO: 63) X-RRRARRRARR(SEQ ID NO: 64) X-RRRPRRRPRR (SEQ ID NO: 65) X-RRPRRPRR (SEQ ID NO: 66)X-RRARRARR (SEQ ID NO: 67)

X can represent a free amino terminus, one or more amino acids, or aconjugated moiety. Internalization peptides can be used in inverso orretro or inverso retro faun with or without the linked peptide orpeptidomimetic being in such form. For example, a preferred chimericpeptide has an amino acid sequence comprising or consisting ofRRRQRRKKRGY-KLSSIESDV (SEQ ID NO:70).

Internalization peptides can be attached to pharmacological agents byconventional methods. For example, the agents can be joined tointernalization peptides by chemical linkage, for instance via acoupling or conjugating agent. Numerous such agents are commerciallyavailable and are reviewed by S. S. Wong, Chemistry of ProteinConjugation and Cross-Linking, CRC Press (1991). Some examples ofcross-linking reagents include J-succinimidyl3-(2-pyridyldithio)propionate (SPDP) or N,N-(1,3-phenylene)bismaleimide;N,N′-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11carbon methylene bridges (which relatively specific for sulfhydrylgroups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversiblelinkages with amino and tyrosine groups). Other cross-linking reagentsinclude p,p′-difluoro-m, m′-dinitrodiphenylsulfone (which formsirreversible cross-linkages with amino and phenolic groups); dimethyladipimidate (which is specific for amino groups);phenol-1,4-disulfonylchloride (which reacts principally with aminogroups); hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate (which reacts principally with amino groups);glutaraldehyde (which reacts with several different side chains) anddisdiazobenzidine (which reacts primarily with tyrosine and histidine).

For pharmacological agents that are peptides attachment to aninternalization peptide can be achieved by generating a fusion proteincomprising the peptide sequence fused, preferably at its N-terminus, toan internalization peptide.

Pharmacologic peptides, optionally fused to tat peptides, can besynthesized by solid phase synthesis or recombinant methods.Peptidomimetics can be synthesized using a variety of procedures andmethodologies described in the scientific and patent literature, e.g.,Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley &Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.

III. Non-Human Primate Model

As discussed in the Background section, several previous clinical trialsof pharmacological agents for treating stroke have failednotwithstanding that the agents have shown promising results in loweranimal models of stroke, such as the rat. Performing similar modelexperiments on non-human primates would provide a better indicator ofhow an agent would perform in humans and thus possibly save the effortand expense of an unsuccessful clinical trial. However, the types ofexperiments performed on rats usually involve sacrificing the animal andwould thus be unethical and/or prohibitively expensive to perform onprimates to the extent necessary to evaluate an agent.

The present application provides a primate model of cerebral ischemicdisease that does not require sacrifice of the primate and results inlittle if any permanent harm to the primate. Cerebral ischemia isinduced by introducing particles into a cerebral blood vessel of aprimate. The particles can be introduced by endovascular surgeryanalogous to that performed on human subjects receiving such surgery fortreatment of aneurysm. The particles used can be about 50-200 andpreferably 75-150 or 100 microns in diameter. The number of particlesintroduced can be e.g., 1-100, or 10-30 with about 20 per animal beingpreferred. The number and size of particles increase the severity of thestroke(s). For example, a particle size of 400 microns can cause strokesso large animals may not recover. Polystyrene microspheres fromPolysciences, Inc. are suitable. Any type of non-human primate, such asmacaque, marmoset, baboon or chimpanzee, can be used in the model. Anyagent can be tested including those described above or previously testedin animal models or clinical trials in the art.

The effect of introducing the particles can be assessed by for example,MRI, CAT scanning and/or behaviorally. MRI analysis shows thatintroducing the particles usually induces several small infarctionsaround the site of the particles. MRI analysis is preferably diffusionweighted (DW-MRI). Diffusion-weighted MRI is a standard technique forimaging stroke in which infarctions appear darker than surroundingtissue. The use of a bipolar gradient pulse and suitable pulse sequencespermits the acquisition of diffusion weighted images (images in whichareas of rapid proton diffusion can be distinguished from areas withslow diffusion). diffusion-weighted images are very helpful to identifyany area of acute ischemia and to separate the acute infarction from oldstrokes and other chronic changes in the brain. Only the acute infarctsappear hyperintense on the diffusion images.

The appearance of the infarction is similar to infarctions often foundin human subjects undergoing endovascular surgery to repair an aneurysm.Infarctions can be assessed, for example, after 4 hr, 24 hr or 14 daysafter introducing the particles. Typically injecting twenty 100 micronmicrospheres produces between 12 and 14 infarcts detectable by MRIimaging. The typical brain volume occupied by such infarcts is about5-10 mm³ each, or at total of about 60-70+/−30 mm³. In human subjectsundergoing endovascular surgery infarcts are larger, typically 5-10 mmin diameter corresponding to about 0.5 to 1.5 cm³ each

A pharmacological agent can be administered to the primate before,during or after introduction of the particles. In some methods, thepharmacological agent is administered within a window of 0-3 or 1-3 hrafter introducing the particles. In other methods, the pharmaceuticalagent is administered 0-3 hours before introducing the particles.Vehicle (e.g., PBS) is usually administered to a control primate alsoreceiving the particles in parallel with administration of thepharmacological agent.

The effects of treatment are assessed by comparing number, volume and/orappearance of infarctions and/or behavioral characteristics between atreated primate or population of treated primates and a control orpopulation of control primates. A reduction in number and/or volume ofinfarctions in the treated primate(s) relative to control primate(s)indicates the agent has activity potentially useful in treating strokeand other diseases mediated in part by excitotoxicity.

A variety of methods have been described for assessing cognitivefunction or neurological damage in non-human primates (see Marshall etal., Stroke 2003; 34:2228-2233; Stroke 2001; 32:190-198; Brain Res Bull2003; 61:577-585; Hatsopoulos et al., Proc Natl Acad Sci USA 1998;95:15706-15711; Spetzler et al., Neurosurgery 1980; 7:257-261; Barbay etal., Exp Brain Res 2006; 169:106-116. An exemplary experiment involvesconditioning monkeys to use their upper limb to visually guide a cursoron a computer screen using planar reaching movements from a centralholding position to a radially located target position. The time to movethe cursor to the target provides a measure of cognitive function orconversely neurological damage.

After analysis of treated and control animals is complete, the animalsare given a period to recover during which the diffusion weighted MRIsignal from the infarctions disappears. This period is typically about10-14 days. After about 14-28 days after initially inserting the beads,the protocol is repeated optionally, with the treated animals serving ascontrols and vice versa. The cross-repetition of the protocol betweentreated and control animal serves to eliminate any bias of the resultsdue to anomalies of particular animals. According to such a repeatedprotocol, reliable results can be obtained from a trial involving onlyten primates. Each primate can undergo at least five cycles of particleinsertion and then continue to live normal lives with little if anypermanent neurological damage.

The experimental model is particularly useful for assessingpharmacological agents for use in treating stroke and for use as anadjunct in method of surgery involving any manipulation of blood vesselssupplying the brain, particularly in endovascular repair of brainaneurysm. The trial also provides a useful indication of the suitabilityof a pharmacological agent for use in treating other diseases anddisorders characterized by cerebral ischemia and/or mediated byexcitotoxity. If a pharmacological agent inhibits development ofinfarctions or neurological behavioral impairment in the primate trial,the agent can be tested in human clinical trials, for example, a trialin stroke subjects, or on subjects undergoing endovascular repair of abrain aneurysm.

IV. Clinical Trial

Procedures that involve manipulating the great vessels, such as theaortic arch (cardiopulmonary bypass), and procedures in which cathetersare introduced into the cervical carotid arteries, such as diagnosticand therapeutic cerebral angiograms, commonly dislodge embolic materialsthat may lead to ischemic strokes of varying frequency and severity(reviewed by Bendszus, Lancet Neurol 2006 April; 5(4):364-72 2006). Forexample, in a representative study of thirty-five consecutive subjectsundergoing elective coronary artery bypass grafting (CABG),diffusion-weighted magnetic resonance imaging demonstrated new ischemiclesions in 9 (26%) of the subjects (Bendszus et al., Arch. Neurol. 59(7):1090-1095, 2002), with such estimates of new lesions bydiffusion-weighted MRI (DWI-MRI) ranging in the literature up to 45% inCABG subjects (Knipp et al., Eur. J. Cardiothorac. Surg. 25 (5):791-800,2004) and 47% in subjects undergoing cardiac valve repairs (Knipp etal., Eur. J. Cardiothorac. Surg. 28 (1):88-96 2005). The majority ofsubjects exhibit two or more lesions (Knipp et al., 2004, supra). Thiseffect is seen in subjects undergoing either on-pump or off-pump CABG,probably because it is the manipulation of the great vessels that iscritical for dislodging damaging emboli.

Cerebral angiography is, in itself, causative of strokes. Bendszus etal. Lancet 354 (9190):1594-1597 (1999) carried out similar studies insubjects undergoing diagnostic or interventional cerebral angiography.

In a prospective study of 100 consecutive angiographies (66 diagnosticand 34 interventional procedures) were done on 91 subjects,diffusion-weighted magnetic resonance imaging (MRI) was performed beforeand after angiography to assess embolic events was performed. Beforeangiography, no abnormalities were seen on diffusion weighted MRI.Diffusion-weighted MRI showed 42 bright lesions in 23 subjects after 23procedures (17 diagnostic, six interventional) in a pattern consistentwith embolic events.

After diagnostic angiography in subjects with a history of vasculopathy,the frequency of lesions was significantly higher than in subjectswithout vascular risk factors (44% vs. 13%, p=0.03). Endovascularaneurysm coiling increases dramatically the risk of procedural strokescompared with diagnostic angiography. In a prospective study, Cronqvistet al., Neuroradiology 47(11):855-73 (2005) demonstrated 47 new ischemicstrokes in 37 of 40 subjects (92.5%) undergoing endovascular aneurysmrepair. In a study performed on 47 subjects undergoingneurointerventional repair of brain aneurysms at the University HealthNetwork, the incidence of small ischemic strokes detected by DWI-MRIimaging was 75%, with most subjects experiencing at least two embolicevents. In the majority of cases, the strokes that occur afterangiography or cardiopulmonary bypass are small-volume round lesions,<20 mm in diameter, with an appearance similar to that of lacunarstrokes. Although they generally do not produce immediate postproceduraldeficits, a significant literature now strongly links small strokesacquired throughout life with deteriorations in cognition, early onsetvascular dementias and Alzheimer's disease (Devasenaphthy and Hachinski,Options Neurol 2:61-72 (2000); Merino and Hachinski, Curr AtherosclerRep 4:285-290 (2002); Di and Hachinski, Curr Opin Investig Drugs4:1082-1087 (2003); Del et al., J Neurol Sci 231:3-11 (2005); Hachinski,Stroke 38:1396 (2007).

Acute ischemic strokes afflict an elderly patient population whocommonly have medical co-morbidities such as ischemic heart disease,diabetes, hypertension, and other disorders whose prevalence rises inlater decades. The patient population is therefore more vulnerable todrug toxicity than is generally the case in other clinical trial.Another difficulty in conducting a clinical trial on patients sufferingfrom acute ischemic strokes is that they must be enrolled in trialswithin a restricted time window, do not uniformly receive invasivemonitoring, and generally are not cared for by anesthesiologists at theoutset. In the past, some clinical trials for acute ischemic stroke havehad to be stopped due to safety issues arising in Phase 3, despiteapparent tolerability of the candidate drug in earlier trials.

Patients who are undergoing surgery for intracranial aneurysms provide asuitable patient population to test the effects of pharmacologicalagents and represent a further unmet medical need. Such patients areusually elderly (the incidence of aneurysm diagnosis peaks in the 6thand 7th decades), thus recapitulating the age-range of the patientpopulation that is most commonly afflicted by acute ischemic stroke. Asdiscussed above, the vast majority develop at least one smallinfarction. Moreover, the standard of care for aneurysm patientsnormally includes constant monitoring by an anesthesiologist, cardiacmonitoring with an ECG, and invasive monitoring, entailing arteriallines for constant blood pressure monitoring and central (right atrial)lines when deemed necessary, constant monitoring of blood oxygensaturation and periodic measurements of arterial blood gases (O₂, PCO₂,pH) and, if needed, serum glucose and electrolytes. Drug administrationcan take place under conditions of endotracheal intubation andartificial ventilation, providing the ultimate safety scenario in theevent of cardio-respiratory compromise, and enabling ultimate control ofall cardiac and ventilatory parameters. The drug administration andrecovery can be done in an environment where the patient have 1:1 careby an anesthesiologist and other medical and paramedical staff,including nursing. The feasibility of increased monitoring and othersafety provisions in a clinical trial provides more opportunity todetect and address any adverse events without terminating the clinicaltrial.

The clinical trial can be performed on any population of patientsundergoing a surgical procedure on a blood vessel supplying the brain(that is connecting the brain to the heart, for example, carotidarteries and jugular veins) or on the brain or CNS itself associatedwith measurable development of infarctions, such as those describedabove. Clinical trials can also be performed on patients undergoing anendovascular procedure on an artery supplying blood to the retina,kidney, spinal cord or limbs. A preferred class of patients are thoseundergoing endovascular surgery to treat a brain aneurysm. Theendovascular surgery can entail for example introducing coils into theaneurysm or a stent into the blood vessel to which the aneurysm isattached. The agent can be administered before, during or after theendovascular surgery is completed. A preferred window for administrationis from 30 minutes before to 1 hour after completing the endovascularsurgery. For example, the pharmacological agent can be administered with0-30 minutes of completion of surgery. The clinical trial is preferablya control trial in which some patients received a pharmacological agentand other patients receive a placebo or vehicle lacking thepharmacological agent. The effects of treatment with the pharmacologicalagent can be assessed by reduction in the number and/or volume ofinfarctions in the treated patients relative to the control population.Infarctions can be assessed in the brain or other tissues, such asretina, kidney, spinal cord and limbs (e.g., by MRI) in comparison withplacebo treated patients. Neurological deficits can also be assessedfrom behavioral characteristics. Pharmacokinetic parameters, such asmaximum serum concentration of the drug under test, serum half-life,serum area under curve, and CSF concentration of the drug can also bemeasured.

Some pharmacological agents tested in such a clinical trial haveundergone previous testing in an animal model of stroke or othercerebral ischemic disease. The animal model can be a lower animal model,such as a rat, or a primate model, such as that discussed above. Someagents have also undergone safety evaluation in a phase I human clinicaltrial such as described in the examples. If a pharmacological agentshows evidence of useful activity in aneurysm patients, the agent can betested in a clinical trial of acute stroke patients or can beadministered to such patients in regular clinical use. The types ofpharmacological agent that can be tested include those described aboveand otherwise tested in the art in animal models of stroke or cerebralischemic disease or in previous clinical trials.

V. Diseases

The pharmacological agents of the invention are useful in treating avariety of diseases, particularly neurological diseases, and especiallydiseases mediated in part by excitotoxity. Such diseases and conditionsinclude stroke, epilepsy, hypoxia, traumatic injury to the CNS notassociated with stroke such as traumatic brain injury and spinal cordinjury, other cerebral ischemia, Alzheimer's disease and Parkinson'sdisease. Other neurological diseases treatable by agents of theinvention not known to be associated with excitotoxicity include anxietyand pain.

A stroke is a condition resulting from impaired blood flow in the CNSregardless of cause. Potential causes include embolism, hemorrhage andthrombosis. Some neuronal cells die immediately as a result of impairedblood flow. These cells release their component molecules includingglutamate, which in turn activates NMDA receptors, which raiseintracellular calcium levels, and intracellular enzyme levels leading tofurther neuronal cell death (the excitotoxicity cascade). The death ofCNS tissue is referred to as infarction. Infarction Volume (i.e., thevolume of dead neuronal cells resulting from stroke in the brain) can beused as an indicator of the extent of pathological damage resulting fromstroke. The symptomatic effect depends both on the volume of aninfarction and where in the brain it is located. Disability index can beused as a measure of symptomatic damage, such as the Rankin StrokeOutcome Scale (Rankin, Scott Med J; 2:200-15 (1957)) and the BarthelIndex. The Rankin Scale is based on assessing directly the globalconditions of a patient as follows.

TABLE 3 0 No symptoms at all 1 No significant disability despitesymptoms; able to carry out all usual duties and activities. 2 Slightdisability; unable to carry out all previous activities but able to lookafter own affairs without assistance. 3 Moderate disability requiringsome help, but able to walk without assistance 4 Moderate to severedisability; unable to walk without assistance and unable to attend toown bodily needs without assistance. 5 Severe disability; bedridden,incontinent, and requiring constant nursing care and attention.

The Barthel Index is based on a series of questions about the patient'sability to carry out 10 basic activities of daily living resulting in ascore between 0 and 100, a lower score indicating more disability(Mahoney et al., Maryland State Medical Journal 14:56-61 (1965)).

Alternatively stroke severity/outcomes can be measured using the NIHstroke scale, available at world wide webninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf.

The scale is based on the ability of a patient to carry out 11 groups offunctions that include assessments of the patient's level ofconsciousness, motor, sensory and language functions.

An ischemic stroke refers more specifically to a type of stroke thatcaused by blockage of blood flow to the brain. The underlying conditionfor this type of blockage is most commonly the development of fattydeposits lining the vessel walls. This condition is calledatherosclerosis. These fatty deposits can cause two types ofobstruction. Cerebral thrombosis refers to a thrombus (blood clot) thatdevelops at the clogged part of the vessel “Cerebral embolism” refersgenerally to a blood clot that forms at another location in thecirculatory system, usually the heart and large arteries of the upperchest and neck. A portion of the blood clot then breaks loose, entersthe bloodstream and travels through the brain's blood vessels until itreaches vessels too small to let it pass. A second important cause ofembolism is an irregular heartbeat, known as arterial fibrillation. Itcreates conditions in which clots can form in the heart, dislodge andtravel to the brain. Additional potential causes of ischemic stroke arehemorrhage, thrombosis, dissection of an artery or vein, a cardiacarrest, shock of any cause including hemorrhage, and iatrogenic causessuch as direct surgical injury to brain blood vessels or vessels leadingto the brain or cardiac surgery. Ischemic stroke accounts for about 83percent of all cases of stroke.

Transient ischemic attacks (TIAs) are minor or warning strokes. In aTIA, conditions indicative of an ischemic stroke are present and thetypical stroke warning signs develop. However, the obstruction (bloodclot) occurs for a short time and tends to resolve itself through normalmechanisms. Patients undergoing heart surgery are at particular risk oftransient cerebral ischemic attack.

Hemorrhagic stroke accounts for about 17 percent of stroke cases. Itresults from a weakened vessel that ruptures and bleeds into thesurrounding brain. The blood accumulates and compresses the surroundingbrain tissue. The two general types of hemorrhagic strokes areintracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic strokeresult from rupture of a weakened blood vessel ruptures. Potentialcauses of rupture from a weakened blood vessel include a hypertensivehemorrhage, in which high blood pressure causes a rupture of a bloodvessel, or another underlying cause of weakened blood vessels such as aruptured brain vascular malformation including a brain aneurysm,arteriovenous malformation (AVM) or cavernous malformation. Hemorrhagicstrokes can also arise from a hemorrhagic transformation of an ischemicstroke which weakens the blood vessels in the infarct, or a hemorrhagefrom primary or metastatic tumors in the CNS which contain abnormallyweak blood vessels. Hemorrhagic stroke can also arise from iatrogeniccauses such as direct surgical injury to a brain blood vessel. Ananeurysm is a ballooning of a weakened region of a blood vessel. If leftuntreated, the aneurysm continues to weaken until it ruptures and bleedsinto the brain. An arteriovenous malformation (AVM) is a cluster ofabnormally formed blood vessels. A cavernous malformation is a venousabnormality that can cause a hemorrhage from weakened venous structures.Any one of these vessels can rupture, also causing bleeding into thebrain. Hemorrhagic stroke can also result from physical trauma.Hemorrhagic stroke in one part of the brain can lead to ischemic strokein another through shortage of blood lost in the hemorrhagic stroke.

One patient class amenable to treatments are patients undergoing asurgical procedure that involves or may involve a blood vessel supplyingthe brain, or otherwise on the brain or CNS. Some examples are patientsundergoing cardiopulmonary bypass, carotid stenting, diagnosticangiography of the brain or coronary arteries of the aortic arch,vascular surgical procedures and neurosurgical procedures. Additionalexamples of such patients are discussed in section IV above. Patientswith a brain aneurysm are particularly suitable. Such patients can betreated by a variety of surgical procedures including clipping theaneurysm to shut off blood, or performing endovascular surgery to blockthe aneurysm with small coils or introduce a stent into a blood vesselfrom which an aneurysm emerges, or inserting a microcatheter.Endovascular procedures are less invasive than clipping an aneurysm andare associated with a better patient outcome but the outcome stillincludes a high incidence of small infarctions. Such patients can betreated with an inhibitor of PSD95 interaction with NMDAR 2B andparticularly the agents described above including the peptideYGRKKRRQRRRKLSSIESDV (SEQ ID NO:6, also known as Tat-NR2B9c). The timingof administration relative to performing surgery can be as describedabove for the clinical trial.

VI. Effective Regimes of Administration

A pharmacological agents optionally linked to an internalization peptideis administered in an amount, frequency and route of administrationeffective to cure, reduce or inhibit further deterioration of at leastone sign or symptom of a disease in a patient having the disease beingtreated. Unless otherwise indicated dosages for chimeric agentsincluding a pharmacologic agent linked to an internalization peptiderefer to the whole agent rather than just the pharmacological agentcomponent of the chimeric agent. A therapeutically effective amountmeans an amount of agent sufficient significantly to cure, reduce orinhibit further deterioration of at least one sign or symptom of thedisease or condition to be treated in a population of patients (oranimal models) suffering from the disease treated with an agent of theinvention relative to the damage in a control population of patients (oranimal models) suffering from that disease or condition who are nottreated with the agent. The amount is also considered therapeuticallyeffective if an individual treated patient achieves an outcome morefavorable than the mean outcome in a control population of comparablepatients not treated by methods of the invention. A therapeuticallyeffective regime involves the administration of a therapeuticallyeffective dose at a frequency and route of administration needed toachieve the intended purpose.

For a patient suffering from stroke or other ischemic condition, theagent is administered in a regime comprising an amount frequency androute of administration effective to reduce the damaging effects ofstroke or other ischemic condition. When the condition requiringtreatment is stroke, the outcome can be determined by infarction volumeor disability index, and a dosage is considered therapeuticallyeffective if an individual treated patient shows a disability of two orless on the Rankin scale and 75 or more on the Barthel scale, or if apopulation of treated patients shows a significantly improved (i.e.,less disability) distribution of scores on a disability scale than acomparable untreated population, see Lees et al., N Engl J Med 2006;354:588-600. A single dose of agent is usually sufficient for treatmentof stroke.

The invention also provides methods and compositions for the prophylaxisof a disorder in a subject at risk of that disorder. Usually such asubject has an increased likelihood of developing the disorder (e.g., acondition, illness, disorder or disease) relative to a controlpopulation. The control population for instance can comprise one or moreindividuals selected at random from the general population (e.g.,matched by age, gender, race and/or ethnicity) who have not beendiagnosed or have a family history of the disorder. A subject can beconsidered at risk for a disorder if a “risk factor” associated withthat disorder is found to be associated with that subject. A risk factorcan include any activity, trait, event or property associated with agiven disorder, for example, through statistical or epidemiologicalstudies on a population of subjects. A subject can thus be classified asbeing at risk for a disorder even if studies identifying the underlyingrisk factors did not include the subject specifically. For example, asubject undergoing heart surgery is at risk of transient cerebralischemic attack because the frequency of transient cerebral ischemicattack is increased in a population of subjects who have undergone heartsurgery as compared to a population of subjects who have not.

Other common risk factors for stroke include age, family history,gender, prior incidence of stroke, transient ischemic attack or heartattack, high blood pressure, smoking, diabetes, carotid or other arterydisease, atrial fibrillation, other heart diseases such as heartdisease, heart failure, dilated cardiomyopathy, heart valve diseaseand/or congenital heart defects; high blood cholesterol, and diets highin saturated fat, trans fat or cholesterol.

A pharmacological agent optionally linked to an internalization peptideis administered to a patient at risk of a disease but not yet having thedisease in an amount, frequency and route sufficient to prevent, delayor inhibit development of at least one sign or symptom of the disease. Aprophylactically effective amount means an amount of agent sufficientsignificantly to prevent, inhibit or delay at least one sign or symptomof the disease in a population of patients (or animal models) at risk ofthe disease relative treated with the agent compared to a controlpopulation of patients (or animal models) at risk of the disease nottreated with a chimeric agent of the invention. The amount is alsoconsidered prophylactically effective if an individual treated patientachieves an outcome more favorable than the mean outcome in a controlpopulation of comparable patients not treated by methods of theinvention. A prophylactically effective regime involves theadministration of a prophylactically effective dose at a frequency androute of administration needed to achieve the intended purpose. Forprophylaxis of stroke in a patient at imminent risk of stroke (e.g., apatient undergoing heart surgery), a single dose of agent is usuallysufficient.

Depending on the agent, administration can be parenteral, intravenous,nasal, oral, subcutaneous, intra-arterial, intracranial, intrathecal,intraperitoneal, topical, intranasal or intramuscular. Intravenousadministration is preferred for peptide agents.

For chimeric agents including an internalization peptide, particularly aHIV tat peptide comprising the amino acid sequence, administration ofthe agent may or may not be combined with an anti-inflammatory agent toreduce release or histamine and its downstream effects associated withhigh levels of the internalization peptide. Preferred agents forco-administration are inhibitors of mast cell degranulation, such ascromolyn or lodoxamide or any others listed herein. Anti-histamines orcorticosteroids can also be used, particularly in combinations or higherdosages (see WO2009/076105, 61/185,943; filed: Jun. 10, 2009 andattorney docket 026373-000920PC filed herewith).

For administration to humans, a preferred dose of the chimeric agentTat-NR2B9c is 2-3 mg/kg and more preferably 2.6 mg/kg. Indicated dosagesshould be understood as including the margin of error inherent in theaccuracy with which dosages can be measured in a typical hospitalsetting. The dose is preferred because it is the maximum dose with whichthe agent can be administered without release of significant amounts ofhistamine and the ensuing sequelae in most patients. Although release ofhistamine at higher dosages can be controlled by co-administration of ananti-inflammatory as discussed above and in any event usuallyspontaneously resolves without adverse events, it is best avoided bykeeping the dose below 3 mg/kg and preferably at 2-3 mg/kg, morepreferably 2.6 mg/kg. Such amounts are for single dose administration,i.e., one dose per episode of disease.

Histamine release is best avoided not only because of well knownsequelae such as reduction in blood pressure, swelling and redness, butbecause of a report that controlling histamine release can also inhibitdevelopment of infarctions in an animal model of stroke (WO 04/071531).Conversely, although lower dosages may well be effective and evenpreferable in indications having a more chronic course (e.g., anxiety,pain, Alzheimer's, Parkinson's) the extremely acute nature of stroke andsimilar conditions may leave very little room for a secondadministration should the first administration prove inadequate orinsufficient. Accordingly, in treating acute presentation, it ispreferably to administer a single dose at or approaching the levelbefore which significant histamine release occurs in most patients.

The dosages indicated above are for the chimeric agent Tat-NR2B9c(YGRKKRRQRRRKLSSIBSDV; SEQ ID NO:6). Equivalent dosages for other agentsto achieve the same effect can be determined by several approaches. Forclose variants of that agent in which one or a few amino acids aresubstituted, inserted or deleted and the molecular weight remains thesame within about +/−25%, the above dosages are still a good guide.However, in general, for other agents, equivalent dosages can varydepending on the molecular weight of the agent with and withoutinternalization peptide if present, its Kd for its target, and itspharmacokinetic and pharmacodynamic parameters. For some agents,equivalent dosages can be calculated so as to deliver an equimolaramount of the pharmacological agent. For other agent, further adjustmentis made to account for differences in Kd or pharmacokinetic orpharmacodynamic parameters. For some agents, equivalent dosages aredetermined empirically from the dose achieved to reach the same endpointin an animal model or a clinical trial.

Peptide agents, such as Tat-NR2B9c are preferably delivered by infusioninto a blood vessel, more preferably by intravenous infusion. The timeof the infusion can affect both side effects (due e.g., to mast celldegranulation and histamine release) and efficacy. In general, for agiven dosage level, a shorter infusion time is more likely to lead tohistamine release. However, a shorter infusion time also may result inimproved efficacy. Although practice of the invention is not dependenton an understanding of mechanism, the latter result can be explainedboth because of the delay being significant relative to development ofpathology in the patient and because of the delay being significantrelative to the plasma half life of the chimeric agent, as a result ofwhich the chimeric agent does not reach an optimal therapeutic level.For the chimeric agent Tat-NR2B9c, a preferred infusion time providing abalance between these considerations is 5-15 minutes and more preferably10 min. Indicated times should be understand as including a marking oferror of +/−10%. Infusion times do not include any extra time for a washout diffusion to wash out any remaining droplets from an initialdiffusion that has otherwise proceeded to completion. The infusion timesfor Tat-NR2B9c can also serve as a guide for other pharmacologicalagents, optionally linked to internalization peptides, particularlyclose variants of Tat-NR2B9c, as discussed above.

VII. Pharmaceutical Compositions

The chimeric or other agents of the invention can be administered in theform of a pharmaceutical composition. Pharmaceutical compositions aretypically manufactured under GMP conditions. Pharmaceutical compositionsfor parenteral administration are preferentially sterile (e.g., filtersterilization of peptide) and free of pyrogens. Pharmaceuticalcompositions can be provided in unit dosage form (i.e., the dosage for asingle administration). Pharmaceutical compositions can be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries that facilitate processingof chimeric agents into preparations which can be used pharmaceutically.Proper formulation is dependent on the route of administration chosen.

An exemplary formulation of the chimeric agent Tat-NR2B9c contains thepeptide in normal saline (0.8-1.0% and preferably 0.9% saline) at aconcentration of 10-30 mg/ml, for example 18-20 mg/ml. When storedfrozen, such a composition is stable (insignificant degradation oraggregation of the peptide) for a period of two or more years. Althoughadditional excipients can be added, normal saline without suchexcipients is sufficient to obtain this stability. For use such acomposition is thawed and diluted into a larger volume of normal salinefor infusion into a blood vessel. Another composition can be made bylyophilizing the chimeric agent Tat-NR2B9c at a concentration of 1-50mg/ml in normal saline in the presence or absence of excipients. Suchexcipients can include those to increase stability or inhibit bacterial,viral or other pathogen growth that could degrade the drug. Thelyophilized composition is stable at −20° C. or at room temperature. Thelyophilized composition can be reconstituted in normal saline.

EXAMPLES Example 1 Primate Model of Ischemic Stroke

The following example was performed on ten macaques divided into fivetest subjects and five controls. Each animal was put through theprotocol twice with test animals in the first round serving as controlsin the second round and vice versa.

Twenty 100-micron polystyrene spheres were delivered into anintracerebral vessel of each animal to induce embolic stroke. One hourafter introducing the sphere, the animals were treated with Tat-NR2B9c(2.6 mg/kg) or vehicle control. The animals were then subject to MRIbrain analysis and neurological examination at 4 hr, 24 hr and 14 daysafter introduction of the spheres.

After 14-28 days, the procedure was repeated with test animals servingas controls and vice versa.

FIG. 1 shows the MRI scan of infarctions 24 hr post injection of spheresin a control animal compared with a human having received endovascularsurgery to insert a coil to repair an aneurysm. The number, size andappearance of infarctions relative to brain size are comparable betweenthe human and the animal.

FIG. 2 compares the number and volume of MRI-visible infarctions intreated and control animals. Treatment with Tat-NR2B9c significantlyreduced the number and volume of infarctions in the whole brain. FIG. 3shows similar data for infarctions in the cortex. The reduction ofnumber and volumes of infarctions was even greater in the cortex thatthe whole brain. Reduction in the cortex is particularly importantbecause this region of the brain is primarily responsible for cognitivefunctioning.

Example 2 Infusion Time

Tat-NR2B9c was administered to rats at 50 mg/kg and infusion times of 3min or 60 min. FIG. 4 shows changes in blood pressure followingadministration. In rats infused over 3 min, the blood pressure decreasedby about 50% before recovering over a period of 90 min. For rats infusedover an hour there was only a slight reduction in blood pressure, whichalso recovered over a period of hour.

Different infusion times were also compared for efficacy in a rat modelof stroke in which adult Sprague Dawley rats (10-12 weeks old) (males˜300 g, females ˜250 g) were fasted for 12-18 hours before beingsubjected to permanent pial vessel occlusion of 3 terminal branches ofthe Middle Cerebral Artery over the Whisker Barrel Cortex (P3VO). Theprotocol for analysis of this rat model has been described inWO/2008/008348. Tat-NR2B9c was administered at 7.6 mg/kg i.v. aftervessel occlusion in comparison with vehicle. Two periods of infusionwere compared, 5 min and 1 hour. FIG. 5 shows infarction size measured24 hr post treatment was significantly reduced relative to salineplacebo with the 5 min infusion but not with the one hour infusion.

Example 3 Phase I Clinical Trial

We carried out a Safety, Tolerability and Pharmacokinetic Study ofTat-NR2B9c in humans. Subjects were either normal, healthy, non-smokingmales or post-menopausal or surgically sterile female subjects with aminimum age of 18 years. The subjects were either administeredTat-NR2B9c, Lot #: 124-134-001B, or were given placebo (PhosphateBuffered Saline), Lot #: 124-134-001A, administered as an intravenousinfusion (10±1 minutes). Four subjects were dosed in each of Cohorts 1to 3, and 10 subjects were dosed in each of Cohorts 4 to 8. All 62subjects completed the study.

Methods Blood Draw Timepoints:

During the study period, 11 blood samples were collected forpharmacokinetic analysis from each subject at the following timepoints:0.00 (pre-dose), 0.08 (5 minutes), 0.17 to 0.25 (10 to 15 minutes,precisely at the end of each individual drug infusion), 0.33 (20minutes), 0.50, 0.75, 1.00, 2.00, 6.00, 12.00, and 24.00 hourspost-dose. In addition, 8 blood samples were collected for histamineanalysis from each subject at the following timepoints: 0.00 (pre-dose),and at 0.08 (5 minutes), 0.17 (10 minutes), 0.25, 0.50, 1.00, 2.00, and24.00 hours post-dose.

Safety Assessment:

The safety assessment was performed on all subjects who received atleast 1 dose during the course of the study. The incidents of alladverse events (AEs) were tabulated by treatment and subject number.Absolute values for vital signs, electrocardiogram (ECG) parameters,laboratory parameters and physical examinations were also documented andvalues outside the normal range were flagged. Shifts from baselinevalues were tabulated. AEs were documented using investigator andMedical Dictionary for Regulatory Activities (MedDRA) terms.

A. Results

Seven of 8 subjects in the 3.75 mg/kg dose group had histamine levelsgreater than 10 nmol/L (average 24.3 nmol/L; maximum of 39.8 nmol/L) 10minutes after the start of NA 1 administration, and 3 of the subjectsstill had histamine levels greater than 10 nmol/L (average 15.3 nmol/L;maximum of 20.3 nmol/L) 15 minutes after the start of NA 1administration.

Other than the 3.75 mg/kg dose group, no treatment group had significantabnormal levels of histamine. The placebo group and the 0.375 mg/kg dosegroup each had 1 subject that had an elevated histamine level at 1timepoint, but these results were at screening and at 2.00 hours postdose, respectively. All abnormal histamine results returned to thenormal range within 24.00 hours of drug administration.

Forty subjects who participated in the study experienced a total of 168adverse effects (AEs) during the study. The majority of AEs were mild inseverity. Thirty-four of 46 active treatment subjects (73.9%)experienced at least 1 AE, while 6 of 16 placebo treatment subjects(37.5%) experienced at least 1 AE. Subjects in the 2.60 and 3.75 mg/kgdose groups experienced significantly more AEs than subjects in thelower dose groups. No Serious Adverse Events (SAEs) were reported. Themost common AEs experienced by subjects receiving Tat-NR2B9c werefeeling hot (13/46; 28.3%), pruritis (12/46; 26.1%), flushing (10/46;21.7%), and dry mouth (9/46; 19.6%). All AEs were resolved with theexception of 2 instances of increased blood glucose, as the subjectswere lost to follow-up.

The incidence of AEs in the 2.60 and 3.75 mg/kg dose groups was higherthan the AE incidence rate in the placebo, 0.02, 0.08, 0.20, 0.375, 0.75and 1.50 mg/kg dose groups. At doses of Tat-NR2B9c>2.60 mg/kg, severalAEs were frequently reported. These included: (1) decreases in bloodpressure, (2) tingling sensation (paraesthesia), (3) numbness(hypoaesthesia), (4) redness (erythema), (5) rash, (6) itchiness(pruritus), (7) dry mouth, (8) nausea, (9) feeling hot, and (10)flushing. The onset of these AEs coincided with the administration ofthe study drug and was probably related to the study drug.

In preclinical trials on rats, dogs and primates, with Tat-NR2B9c,elevated histamine levels were observed in high dose groups, and werelikely the source of side effects including swelling, redness andhypotension. In the current study, histamine levels were elevated in 7of the 8 subjects in the highest dose group (3.75 mg/kg) 10 minutesafter the start of the intravenous drug administration, and remainedelevated in 3 of these subjects 15 minutes after drug administration,after which time levels returned to the normal range. During the sametime frame that histamine levels were elevated, most of the AEs in the3.75 mg/kg dose group were observed. This suggests that the elevatedhistamine levels were the source of the most frequently reported AEs(including decreased blood pressure, tingling, numbness, redness, rash,itchiness, dry mouth, nausea, feeling hot, and flushing).

Example 4 Clinical Trial of Tat-NR2B9c in Patients UndergoingNeurointerventional Aneurysm Repair Procedures

The primary measure of efficacy is the total volume of MRI-detectableDWI and FLAIR-sequence-positive lesions following endovascularintervention. If there are a number of patients with large volumestroke, these outlier volumes are truncated to a maximum of 10 cc toallow for a more normal distribution of volumes and testing withstandard parametric statistics. The primary analysis is a t-test, with anull hypothesis of equivalent means in the two treatment groups (drugvs. Placebo) and an alternative hypothesis that the mean lesion totalvolume in the treatment group is smaller than the placebo group. Themaximum sample size of the trial is 400, with 200 per treatment arm.

A determination of the efficacy of a single intravenous dose of agent inreducing the number of embolic strokes induced by the endovasculartreatment is based on counts of strokes from the DWI/FLAIR MRIsequences. The number of strokes per patient in each of the treated andplacebo groups is sorted into five categories as follows: 0 (nostrokes), 1, 2, 3, and 4 or more strokes.

The ability of the agent to reduce procedurally-induced vascularcognitive impairment is determined using a weighted composite scoreobtained from the neurocognitive test Battery.

To determine the efficacy of the agent in reducing the frequency oflarge (>10 cc volume) strokes, the number of large strokes in thetreated and placebo group is compared using a contingency table analysisas above.

Modified Rankin Scores (mRS; range, 0 to 5, with 0 indicating noresidual symptoms and 5 indicating bed-bound, requiring constant care)in patients undergoing the endovascular procedure are dichotomized intomRS 0 to 2 (indicating independent functioning) and mRS of 3 or greater,indicating death or dependency. The NIH stroke scale (NIHSS; scoresrange from 0 to 42, with higher scores indicating increasing severity)data are assessed as a dichotomy: NIHSS 0-1 vs. 2 or greater using aChi-square or Fisher's exact test. The analysis is further stratifiedaccording to whether or not the patient presented with a subarachnoidhemorrhage.

Dosing begins as soon as the endovascular repair of the brain aneurysmhas been completed (typically after insertion of the final coil orstent) and the final angiographic imaging has been completed. Dosetiming starts at the time of drip onset. Dosing is performed byadministering the contents of the 100 mL bag to the subject through anintravenous catheter inserted into a vein in the upper extremity andusing an infusion pump [e.g., FLO-GARD Volumetric Infusion Pump 6201 orequivalent]. Dosing is carried out evenly over the course of 10±1minutes while the IV bag contents are administered to the subject. Theentire volume (treatment dose) of the IV mini-bag is administered. Afterthe dose administration, a minimum of 10 mL (not to exceed 15 mL) ofsaline is administered using the infusion pump to flush any remainingmedication left within the IV tubing.

Example 5 Dose Preparation

Syringe vials containing Tat-NR2B9c in normal (0.9%) saline or placeboare stored at the pharmacy of each clinical site at −20° C. On enrolmentof a study subject, one or more (depending on the total required)Tat-NR2B9c drug or placebo syringe vial is elected at the pharmacy basedupon a drug:placebo randomization code and thawed before use. A syringefor each individual subject dosing is labeled with the subject numberand prepared by calculating the volume to draw from the syringe vial asfollows: (2.60 mg/kg×subject weight in kg)/[Drug Potency in mg/ml]. Thisdetermines the number of millilitres to pull up into the syringe. Thesyringe containing Tat-NR2B9c or placebo is delivered to the dosing siteand there injected into the IV port of a 100 mL drip bag of 0.9% normalsaline.

Although the invention has been described in detail for purposes ofclarity of understanding, it will be obvious that certain modificationsmay be practiced within the scope of the appended claims. Allpublications, accession numbers, and patent documents cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each were so individually denoted.To the extent more than one sequence is associated with an accessionnumber at different times, the sequences associated with the accessionnumber as of the effective filing date of this application is meant. Theeffective filing date is the date of the earliest priority applicationdisclosing the accession number in question. Unless otherwise apparentfrom the context any element, embodiment, step, feature or aspect of theinvention can be performed in combination with any other.

1. A method of treating or effecting prophylaxis of disease mediated byexcitotoxicity, comprising administering to a subject having or at riskof the disease a pharmacological agent that inhibits binding of PSD-95to NMDAR 2B and/or of PSD-95 to nNOS, wherein when the agent is apeptide having the amino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ IDNO:6), the dose is 2-3 mg/kg, and if the agent is other than the peptidehaving the amino acid sequence YGRKKRRQRRRKLSSIESDV, the dose deliversthe equivalent effective concentration of the agent to 2-3 mg/kg of thepeptide having the amino acid sequence YGRKKRRQRRRKLSSTESDV.
 2. Themethod of claim 1, wherein the agent is the peptide having the aminoacid sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6) and the dose is 2.6mg/kg.
 3. The method of claim 1, wherein the dose is administered onceper episode of the disease.
 4. The method of claim 1, where the dose isadministered without-co-administration of an anti-inflammatory agent. 5.A method of treating or effecting prophylaxis of a disease mediated byexcitotoxicity, comprising administering to a subject having or at riskof the disease a pharmacological agent that inhibits binding of PSD-95to NMDAR 2B and/or PSD-95 to nNOS, wherein the agent is linked to aninternalization peptide, and the agent is administered by intravenousinfusion over a period of 5-15 minutes.
 6. The method of claim 5,wherein the agent linked to the internationalization peptide is thepeptide having the amino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ IDNO:6) and the period is 5 minutes.
 7. The method of claim 6, wherein thedose of the pharmacological agent is greater than 1 mg/kg.
 8. The methodof claim 6, wherein the dose of the pharmacological agent is 2-3 mg/kg.9. The method of claim 6, wherein the dose of the pharmacological agentis 2.6 mg/kg.
 10. The method of claim 6, wherein the dose is up to 50mg/kg, provided that if the dose is greater than 3 mg/kg the dose isco-administered with an anti-inflammatory.
 11. The method of claim 5,wherein the dose is administered without co-administration of ananti-inflammatory agent.
 12. The method of claim 5, wherein the subjecthas a stroke.
 13. The method of claim 5, wherein the subject isundergoing surgery.
 14. The method of claim 13, wherein the subject isundergoing endovascular surgery to treat an aneurysm.
 15. A method ofinhibiting ischemic damage from neurosurgery or endovascular surgery toa blood vessel connecting the brain to the heart or supplying blood to alimb, kidney, retina or spinal cord comprising administering aneffective regime of an agent that inhibits binding of PSD-95 to NMDAR 2Bto a subject undergoing the neurosurgery or endovascular surgery to ablood vessel.
 16. The method of claim 15, wherein ischemic damage isfrom neurosurgery and the neurosurgery is diagnostic angiography of thebrain.
 17. The method of claim 15, wherein ischemic damage is fromneurosurgery and the neurosurgery is endovascular surgery to treat ananeurysm.
 18. The method of claim 15, wherein the agent is administeredbefore the endovascular surgery.
 19. The method of claim 15, wherein theagent is administered within 1 hour of completing endovascular surgery.20. The method of claim 15, wherein the endovascular surgery comprisinginserting a coil into the aneurysm.
 21. The method of claim 15, whereinthe endovascular surgery comprises inserting a stent into the vesselsubject to the aneurysm.
 22. The method of claim 15, wherein theendovascular surgery comprises inserting a microcatheter.
 23. A methodof performing a clinical trial on a pharmacological agent comprisingadministering the pharmacological agent to a population of subjectsundergoing endovascular surgery on a blood vessel in the brain orconnecting the brain to the heart or supplying blood to a limb, retina,kidney or spinal cord; and comparing the frequency of a damaging effectof the surgery in the subjects compared with control subjects undergoingthe endovascular surgery without the pharmacological agent, to determinewhether the pharmacological agent reduces the damaging effect.
 24. Themethod of claim 23, wherein the subjects are undergoing endovascularsurgery for a brain aneurysm.
 25. The method of claim 23, wherein thedamaging effect is assessed by the number and/or size of cerebralinfarctions.
 26. The method of claim 23, wherein the damaging effect isassessed by number and/or size of infarctions in the retina, kidney,spinal cord or limbs.
 27. The method of claim 23, further comprisingtesting the pharmacological agent in an animal model of stroke.
 28. Themethod of claim 23, further comprising testing the pharmacological agentin a human subject having a stroke.
 29. The method of claim 23, furthercomprising labeling the pharmacological agent for treatment of stroke.30. The method of claim 23, wherein the pharmacological agent inhibitsbinding of PSD95 to NMDAR 2B and/or PSD-95 to nNOS.
 31. A method oftesting a pharmacological agent comprising, comprising a. insertingparticles into a cerebral blood vessel of a primate; b. administeringthe pharmacological agent to the primate c. comparing a damaging effectof step (a) in the primate compared to a control primate not treatedwith the compound.
 32. The method of claim 31, wherein the damagingeffect is assessed by number and/or size of infarctions.
 33. The methodof claim 31, wherein the infarctions are determined by MRI or CATscanning.
 34. The method of claim 31, wherein the damaging effect isassessed by a behavioral symptom of the primate compared with thecontrol primate.
 35. The method of claim 31, further comprisingrepeating steps (a)-(c) on the same primate after a recovery periodprovided the agent tested on repeating steps (a)-(c) may or may not bethe same as when steps (a)-(c) were previously preformed.
 36. The methodof claim 31, further comprising repeating steps (a)-(c) except that thecontrol primate receives the agent and the animal previously receivingthe agent is the control animal.
 37. The method of claim 31, wherein theagent inhibits binding of PSD-95 to NMDAR 2B and/or PSD-95 to nNOS. 38.The method of claim 31, further comprising administering the agent to ahuman stroke subject.
 39. The method of claim 31, further comprisingadministering the agent to a human subject undergoing endovascularsurgery for an aneurysm.
 40. The method of claim 31, wherein the agentis administered after inserting particles into the cerebral bloodvessel.
 41. The method of claim 31, wherein the agent is administeredbefore inserting particles into the cerebral blood vessel.
 42. Themethod of claim 31, wherein the particles are 100 micron polystyrenespheres.
 43. The method of claim 42, wherein 20 spheres are administeredto the primate.
 44. The method of claim 31, wherein the primate is amacaque.
 45. A lyophilized composition comprising the peptide having theamino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6) lyophilized froma solution of the peptide in normal saline prepared under GMPconditions.
 46. A method of storing a pharmaceutical compositioncomprising storing a pharmaceutical composition comprising a peptidehaving the amino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6) atconcentration of 10-30 mg/ml in normal saline under GMP conditions atbelow 0° C. for a period of at least two years.
 47. The method of claim46, wherein the concentration is 18-20 mg/ml.
 48. The method of claim 46or 47, further comprising administering the pharmaceutical compositionto a subject.